The present invention relates to a method and an apparatus for frequency thermal control, especially to a method and an apparatus for controlling the temperature of a microfluid by applying the microfluid to a heater at a determined frequency.
The xe2x80x9cpolymerase chain reaction (PCR)xe2x80x9d is a biochemical reaction invented by Kary Mullis in 1985. This technique enables us to produce enormous numbers of copies of specified DNA sequence. PCR utilizes DNA""s double helix structure and base pairing. In PCR, particular base sequences on the order of hundreds of base pairs may be precisely selected from a molecule having millions of base pairs, and duplicated to obtain more than one million copies of such sequences.
In conducting a PCR, three basic steps are necessary:
1. Denature: The double-stranded DNA templates can be separated by heating the reaction mixture to temperatures near boiling.
2. Annealing: While reducing the temperature of the mixture to 30-65, primers anneal to the two primer binding sites that flank the target region, one on each strand.
3. Extension: The temperature of the reaction mixture is again increased to 65-75xc2x0 C., such that the Taq polymerase synthesizes new strands of DNA, complementary to the template, that extend a variable distance beyond the position of the primer binding site on the other template.
When the above-said three steps are conducted repeatedly, the polymerase chain reaction may be conducted. In theory, when n cycles are completed, 2n amplicons may be obtained. For example, if the steps are repeated 20 times, 268,435,456 double helix amplicons may be obtained. However, in practice, the yield rate may be expected according to the following equation:
Yield=(1+e)n, wherein e represents efficiency. In general case, the efficiency will decrease when more than 20 cycles are conducted, in which the efficiency is about 0.8.
There are two major reasons why the efficiency will decrease. The first reason is mismatch of the primers. Even when the position of the primer matched to the template is wrong, the PCR mechanism continues to produce non-targeted amplicons regardless of the mismatch. The product so obtained is call xe2x80x9cugly little fragmentxe2x80x9d or xe2x80x9cprimer dimmerxe2x80x9d which competitively consumes the reaction materials in the mixture. The second possible problem affecting the efficiency of the PCR is damage caused to the polymerase by the repeated denature temperature.
The above problems may be solved by two approaches. The first solution is to create accurate and sharp turning points of the thermocycles. In order to avoid damage to the polymerase due to the continuous high denature temperature and mismatches of the primer due to the low temperature during annealing, optimal denaturation and annealing times are less than 1 sec. The second possible solution is to accelerate the increase and decrease of the temperature. The rapid temperature transitions decrease the amount of nonspecific product. Rapid cooling after denaturation of the DNA template and primer pairs favors the kinetics of specific primer annealing. Besides, maintaining the mixture in a relatively high temperature would damage the polymerase.
U.S. Pat. No. 5,475,610 related to a thermal cycler. The spirit of this patent rested in the interface between its reaction tube and its temperature programmed sample block The reaction tube used in this patent has a thin wall made of polyethylene, The reaction tubes are first plugged into the wells positioned in a metal block and are sealed with upper heated covers. In this patent, the heat exchange area and efficiency may be enhanced such that the volume to surface ratio of the system may reach 0.66 ul/mm2, whereby each 1 mm2 thermal transmission area can support the heat exchange of 0.66 ul PCR samples. A PCR system according to this invention is able to provide a temperature increase speed of about 5xc2x0 C./s. However, the physical contact heating process is used in this patent wherein the metal block is first heated such that the wall of the reaction tube may be heated and that the PCR mixture may be heated. As a result, the total thermal mass of the system is so great that rapid temperature variation is not possible in this system.
If one observes from the view point of temperature variation of the PCR system, the variation of temperature during the process may be divided into four steps: heating, cooling, heating and thermostatic. J L Danssaert et al. disclosed a PCR device comprising four temperature-controlling gradient blocks in their U.S. Pat. No. 5,525,300. In the Danssaert et al. invention, the first and third blocks have respective heaters to raise the temperature, the fourth block has a cooler to drop the temperature and the second block has a heater and a cooler to maintain the temperature. During the operation of the PCR system, a robot is used to remove the test tubes from one block to another. As a result, all test tubes may be subject to heating, cooling, heating and thermostatic steps.
The advantages of the Danssaert et al. invention include that the system may have very short transient state, since the temperatures of the four blocks are already settled, and that rapid temperature variations of the reaction mixture is made possible by the mechanical action with a robot. However, the disadvantages of this invention rest in the interface between the test tubes and the temperature controlling blocks. Because it is necessary to remove the test tubes from time to time, it is impossible to provide tight contacts between the test tubes and the blocks. Heat exchange efficiency of the system is thus reduced. In addition to that, the frequent movement of the robot arm makes the reliability of the system relatively low.
Andreas Manz et al. disclosed a continuous-flow PCR system in 1998. See: Kopp M U, de Mello A J, Manz A, Chemical amplification: continuous-flow PCR on a chip, Science, Vol. 280, pp. 1046-1048, 1998. In this continuous-flow PCR system, a single microchannel passes repetitively through three temperature zones. The microchannel with a cross-section area of 40 xcexcmx90 xcexcm and a length of 2.2 m is etched in a glass chip with the micromachining technology. The pattern of the chip layout determines the relative time in which a fluid element is exposed to each temperature zone. The glass chip is then positioned on an arrangement of three spatially separated copper blocks, with their temperature maintained stably at 60xc2x0 C., 77xc2x0 C. and 95xc2x0 C. respectively. The PCR reaction mixture is supplied to and pumped through the microchannel such that 20 identical thermal cycles, each having a time ratio of 4:4:9 (denature, annealing, extension) may be achieved.
This continuous-flow system provides an advantageous volume to surface ratio of 0.04 xcexc/mm2, about only one seventeenth to that of the conventional PE reaction tube. As a result, when the reactants flow through a copper block, the expanded heat exchange surface enables the temperature of the reactants to reach the same temperature as the surroundings. Another achievement rests in that the continuous xe2x80x9ctimexe2x80x9d in the denature, annealing and extension steps are converted into the accurately defined xe2x80x9cspacexe2x80x9d (the 4:4:9 length proportions) as prepared by the micromachining technology. According to the above-mentioned article, if the flow speed of the reactants is 72.9 xcexcl/s, it takes only 90 seconds to complete 20 cycles. However, such a speed will bring up a new question: the yield of the reaction. According to this article, When the flow speed is 5.8 xcexcl/s, it takes 18.8 minutes to complete 20 cycles and the yield rate could reach about 80% of that of the conventional art. When the flow speed is accelerated, the yield rate will decrease significantly. Obviously when the reaction is completed within 90 seconds, the yield rate should be very low. In addition, as the cycle time ratio of 4:4:9 has been defined by the chip layout, the freedom of adjusting the proportions is thus limited. As a matter of fact, it is always necessary to adjust the cycle time ratio in the practical application of PCR.
It is thus a need in the industry to provide a novel method for thermal control of microfluid that can provide efficient and accurate thermal control.
The objective of this invention is to provide a novel method and device for thermal control of microfluid.
Another objective of this invention is to provide a method and device for frequency thermal control of microfluid.
Another objective of this invention is to provide a method for dynamic, rapid and accurate control of the temperature of a microfluid.
Another objective of this invention is to provide a low-cost device for thermal control of microfluid.
According to this invention a method for thermal control of microfluid and a device using the thermal control method are disclosed. The device of this invention comprises a chip provided with a microchannel and a pneumatic microflow driving element to drive a microfluid to flow back and forth in said microchannel at a predetermined frequency. Heating device are arranged along specific sections of the microchannel. By controlling the frequency of the back-and-forth movement of the microfluid, the temperature of the microfluid may be accurately controlled. Due to the rapid movement of the microfluid in the microchannel, uniform distribution of temperature and ingredients in the microfluid may also be obtained.